The principal objective of this proposal is to extend our studies of Ca2+ homeostasis in resting and stimulated pancreatic acinar cells. This constitutes an essential stage towards understanding the mechanisms of release and reloading of intracellular Ca2+ stores during [Ca2+]i oscillation, and upon termination of cell stimulation. Over the last few years we have developed techniques: to specifically label the agonist-- mobilizable intracellular pool with (45)Ca in intact cells; to measure (45)Ca fluxes mediated by conductive pathways in intact and permeabilized cells and Ca2+ pump-mediated-(45)Ca fluxes in permeabilized cells; to measure free cytosolic Ca 2+ concentration with Fura 2 in cell suspension and in single pancreatic acinus. We have also identified the presence of a cytosolic Ca2+ binding protein which inhibits IP3-mediated Ca 2+ release. Using these techniques the immediate aims of the proposal are to 1) determine the route of Ca 2+ entry across the plasma membrane during stimulation and reloading. This should reveal the role of the cytosolic Ca 2+ pool in controlling reloading of intracellular Ca 2+ stores, 2) characterize [Ca2+]i oscillation of pancreatic acinus and single cells within the acinus in an attempt to determine the role of pool Ca 2+ content and [Ca2+]i in regulating Ca2+ oscillation, 3) define the activation of the intracellular Ca2+ pump by agonists and protein kinases, 4) study the overall regulation of IP3 binding and IP3-mediated Ca 2+ release by the cytosolic inhibitory Ca 2+ binding protein, 5) purify the inhibitory protein and characterize its interaction with the IP3-mobilizable Ca 2+ pool and its distribution in various cells. Regulation of the interaction of this protein with the IP3-activated Ca 2+ channel has the potential of providing a temporal aspect to regulation of IP3-mediated Ca 2+ release and thus resulting in Ca2+ oscillation. We hope that our basic studies are providing techniques to test hypotheses on hormonal regulation of cellular Ca2+ pathways controlling free cytosolic Ca 2+ and will bring us closer toward understanding Ca 2+ homeostasis and Ca 2+ oscillation by the stimulated pancreatic acinar cell.